Introduction
Many compounds absorb ultraviolet (UV) or visible (Vis.) light. The diagram below shows a beam
of monochromatic radiation of radiant power P0, directed at a sample solution. Absorption takes
place and the beam of radiation leaving the sample has radiant power P.
The amount of radiation absorbed may be measured
in a number of ways:
Transmittance, T = P / P0
% Transmittance, %T = 100 T
Absorbance,
A = log10 P0 / P
A = log10 1 / T
A = log10 100 / %T
A = 2 - log10 %T
The last equation, A = 2 - log10 %T , is worth remembering because it allows you to easily calculate
absorbance from percentage transmittance data.
The relationship between absorbance and transmittance is illustrated in the following diagram:
So, if all the light passes through a solution without any absorption, then absorbance is zero, and
percent transmittance is 100%. If all the light is absorbed, then percent transmittance is zero, and
absorption is infinite.
The Beer-Lambert Law
Now let us look at the Beer-Lambert law and explore it's significance. This is important because
people who use the law often don't understand it - even though the equation representing the law is
so straightforward:
A=abc
Where A is absorbance (no units, since A = log10 P0 / P )
ð
a is the molar absorbtivity with units of L mol-1 cm-1
b is the path length of the sample - that is, the path length of the cuvette in which the sample is contained. We will express this measurement in
centimetres.
c is the concentration of the compound in solution, expressed in mol L-1
The reason why we prefer to express the law with this equation is because absorbance is directly
proportional to the other parameters, as long as the law is obeyed. We are not going to deal with
deviations from the law.
Let's have a look at a few questions...
Question : Why do we prefer to express the Beer-Lambert law using absorbance as a measure of
the absorption rather than %T ?
Answer : To begin, let's think about the equations...
A=abc
%T = 100 P/P0 = e -abc
Now, suppose we have a solution of copper sulphate (which appears blue because it has an
absorption maximum at 600 nm). We look at the way in which the intensity of the light (radiant
power) changes as it passes through the solution in a 1 cm cuvette. We will look at the reduction
every 0.2 cm as shown in the diagram below. The Law says that the fraction of the light
absorbed by each layer of solution is the same. For our illustration, we will suppose that this
fraction is 0.5 for each 0.2 cm "layer" and calculate the following data:
Path length / cm 0 0.2 0.4 0.6 0.8 1.0
%T 100 50 25 12.5 6.25 3.125
Absorbance 0 0.3 0.6 0.9 1.2 1.5
A=abc
tells us that absorbance depends on the total quantity of the absorbing compound in the light path
through the cuvette. If we plot absorbance against concentration, we get a straight line passing
through the origin (0,0).
Note that the Law is not obeyed
at high concentrations. This
deviation from the Law is not
dealt with here.
The linear relationship between concentration and absorbance is both simple and straightforward,
which is why we prefer to express the Beer-Lambert law using absorbance as a measure of the
absorption rather than %T.
Question : What is the significance of the molar absorbtivity, a?
ð
Answer : To begin we will rearrange the equation A = ðabc :
ð
a = A / bc
In words, this relationship can be stated as " ð ð ais a measure of the amount of light absorbed per unit
concentration".
Molar absorbtivity is a constant for a particular substance, so if the concentration of the solution is
halved so is the absorbance, which is exactly what you would expect.
Question : What is the molar absorbtivity of Cu2+ ions in an aqueous solution of CuSO4 ? It is
either 20 or 100,000 L mol-1 cm-1
Answer : I am guessing that you think the higher value is correct, because copper sulphate
solutions you have seen are usually a beautiful bright blue colour. However, the actual molar
absorbtivity value is 20 L mol-1 cm-1 ! The bright blue colour is seen because the concentration of
the solution is very high.
-carotene is an organic compound found in vegetables and is responsible for the colour of carrots.
It is found at exceedingly low concentrations. You may not be surprised to learn that the molar
absorbtivity of -carotene is 100,000 L mol-1 cm-1 !
Review your learning
You should now have a good understanding of the Beer-Lambert Law; the different ways in which
we can report absorption, and how they relate to each other. You should also understand the
importance of molar absorbtivity.
Theoretical principles
Introduction
Different molecules absorb radiation of different wavelengths. An absorption spectrum will show a
number of absorption bands corresponding to structural groups within the molecule. For example,
the absorption that is observed in the UV region for the carbonyl group in acetone is of the same
wavelength as the absorption from the carbonyl group in diethyl ketone.
Electronic transitions
The absorption of UV or visible radiation corresponds to the excitation of outer electrons. There are
three types of electronic transition which can be considered.
When an atom or molecule absorbs energy, electrons are promoted from their ground state to an
excited state. In a molecule, the atoms can rotate and vibrate with respect to each other. These
vibrations and rotations also have discrete energy levels, which can be considered as being packed
on top of each electronic level.
Absorption of ultraviolet and visible radiation in organic molecules is restricted to certain functional
groups (chromophores) that contain valence electrons of low excitation energy. The spectrum of a
molecule containing these chromophores is complex. This is because the superposition of rotational
and vibrational transitions on the electronic transitions gives a combination of overlapping lines.
This appears as a continuous absorption band.
Charge - Transfer Absorption
Many inorganic species show charge-transfer absorption and are called charge-transfer complexes.
For a complex to demonstrate charge-transfer behaviour, one of its components must have electron
donating properties and another component must be able to accept electrons. Absorption of
radiation then involves the transfer of an electron from the donor to an orbital associated with the
acceptor.
Molar absorbtivities from charge-transfer absorption are large (greater that 10,000 L mol-1 cm-1).
Review your learning
You should now be aware of why molecules absorb radiation in the UV and visible light regions,
and why absorption spectra look the way they do.
Instrumentation
Introduction
Have a look at this schematic diagram of a double-beam UV-Vis. spectrophotometer;
Instruments for measuring the absorption of U.V. or visible radiation are made up of the following
components;
1. Sources (UV and visible)
2. Wavelength selector (monochromator)
3. Sample containers
4. Detector
5. Signal processor and readout
Each of these components will be considered in turn.
Instrumental components
Sources of UV radiation
It is important that the power of the radiation source does not change abruptly over it's wavelength
range.
The electrical excitation of deuterium or hydrogen at low pressure produces a continuous UV
spectrum. The mechanism for this involves formation of an excited molecular species, which breaks
up to give two atomic species and an ultraviolet photon. This can be shown as;
D2 + electrical energy D2* D' + D'' + hv
Both deuterium and hydrogen lamps emit radiation in the range 160 - 375 nm. Quartz windows
must be used in these lamps, and quartz cuvettes must be used, because glass absorbs radiation of
wavelengths less than 350 nm.
Sources of visible radiation
The tungsten filament lamp is commonly employed as a source of visible light. This type of lamp is
used in the wavelength range of 350 - 2500 nm. The energy emitted by a tungsten filament lamp is
proportional to the fourth power of the operating voltage. This means that for the energy output to
be stable, the voltage to the lamp must be very stable indeed. Electronic voltage regulators or
constant-voltage transformers are used to ensure this stability.
Tungsten/halogen lamps contain a small amount of iodine in a quartz "envelope" which also
contains the tungsten filament. The iodine reacts with gaseous tungsten, formed by sublimation,
producing the volatile compound WI2. When molecules of WI2 hit the filament they decompose,
redepositing tungsten back on the filament. The lifetime of a tungsten/halogen lamp is
approximately double that of an ordinary tungsten filament lamp. Tungsten/halogen lamps are very
efficient, and their output extends well into the ultra-violet. They are used in many modern
spectrophotometers.
Wavelength selector (monochromator)
All monochromators contain the following component parts;
" An entrance slit
" A collimating lens
" A dispersing device (usually a prism or a grating)
" A focusing lens
" An exit slit
Polychromatic radiation (radiation of more than one wavelength) enters the monochromator through
the entrance slit. The beam is collimated, and then strikes the dispersing element at an angle. The
beam is split into its component wavelengths by the grating or prism. By moving the dispersing
element or the exit slit, radiation of only a particular wavelength leaves the monochromator through
the exit slit.
Czerney-Turner grating monochromator
Cuvettes
The containers for the sample and reference solution must be transparent to the radiation which will
pass through them. Quartz or fused silica cuvettes are required for spectroscopy in the UV region.
These cells are also transparent in the visible region. Silicate glasses can be used for the
manufacture of cuvettes for use between 350 and 2000 nm.
Detectors
The photomultiplier tube is a commonly used detector in UV-Vis spectroscopy. It consists of a
photoemissive cathode (a cathode which emits electrons when struck by photons of radiation),
several dynodes (which emit several electrons for each electron striking them) and an anode.
A photon of radiation entering the tube strikes the cathode, causing the emission of several
electrons. These electrons are accelerated towards the first dynode (which is 90V more positive than
the cathode). The electrons strike the first dynode, causing the emission of several electrons for
each incident electron. These electrons are then accelerated towards the second dynode, to produce
more electrons which are accelerated towards dynode three and so on. Eventually, the electrons are
collected at the anode. By this time, each original photon has produced 106 - 107 electrons. The
resulting current is amplified and measured.
Photomultipliers are very sensitive to UV and visible radiation. They have fast response times.
Intense light damages photomultipliers; they are limited to measuring low power radiation.
Cross section of a photomultiplier tube
The linear photodiode array is an example of a multichannel photon detector. These detectors are
capable of measuring all elements of a beam of dispersed radiation simultaneously.
A linear photodiode array comprises many small silicon photodiodes formed on a single silicon
chip. There can be between 64 to 4096 sensor elements on a chip, the most common being 1024
photodiodes. For each diode, there is also a storage capacitor and a switch. The individual diode-
capacitor circuits can be sequentially scanned.
In use, the photodiode array is positioned at the focal plane of the monochromator (after the
dispersing element) such that the spectrum falls on the diode array. They are useful for recording
UV-Vis. absorption spectra of samples that are rapidly passing through a sample flow cell, such as
in an HPLC detector.
Charge-Coupled Devices (CCDs) are similar to diode array detectors, but instead of diodes, they
consist of an array of photocapacitors.
Review your learning
You should now have an understanding of the separate components which make up a
spectrophotometer, and how they fit together. Have a look at this schematic of the Hitachi 100-60
manual double-beam spectrophotometer;
Do you understand what each component does?
Theoretical Principles
Introduction
The term "infra red" covers the range of the electromagnetic spectrum between 0.78 and 1000 m.
In the context of infra red spectroscopy, wavelength is measured in "wavenumbers", which have the
units cm-1.
wavenumber = 1 / wavelength in centimeters
It is useful to divide the infra red region into three sections; near, mid and far infra red;
Region Wavelength range ( m) Wavenumber range (cm-1)
Near 0.78 - 2.5 12800 - 4000
Middle 2.5 - 50 4000 - 200
Far 50 -1000 200 - 10
The most useful I.R. region lies between 4000 - 670cm-1.
Theory of infra red absorption
IR radiation does not have enough energy to induce electronic transitions as seen with UV.
Absorption of IR is restricted to compounds with small energy differences in the possible
vibrational and rotational states.
For a molecule to absorb IR, the vibrations or rotations within a molecule must cause a net change
in the dipole moment of the molecule. The alternating electrical field of the radiation (remember
that electromagnetic radation consists of an oscillating electrical field and an oscillating magnetic
field, perpendicular to each other) interacts with fluctuations in the dipole moment of the molecule.
If the frequency of the radiation matches the vibrational frequency of the molecule then radiation
will be absorbed, causing a change in the amplitude of molecular vibration.
Molecular rotations
Rotational transitions are of little use to the spectroscopist. Rotational levels are quantized, and
absorption of IR by gases yields line spectra. However, in liquids or solids, these lines broaden into
a continuum due to molecular collisions and other interactions.
Molecular vibrations
The positions of atoms in a molecules are not fixed; they are subject to a number of different
vibrations. Vibrations fall into the two main categories of stretching and bending.
Stretching: Change in inter-atomic distance along bond axis
Bending: Change in angle between two bonds. There are four types of bend:
" Rocking
" Scissoring
" Wagging
" Twisting
Vibrational coupling
In addition to the vibrations mentioned above, interaction between vibrations can occur (coupling)
if the vibrating bonds are joined to a single, central atom. Vibrational coupling is influenced by a
number of factors;
" Strong coupling of stretching vibrations occurs when there is a common atom between the
two vibrating bonds
" Coupling of bending vibrations occurs when there is a common bond between vibrating
groups
" Coupling between a stretching vibration and a bending vibration occurs if the stretching
bond is one side of an angle varied by bending vibration
" Coupling is greatest when the coupled groups have approximately equal energies
" No coupling is seen between groups separated by two or more bonds
Review your learning
You should now understand how certain molecules absorb infra red radiation, and the effects that
this absorption has. You should be familiar with the ways in which molecules can vibrate, and
factors which influence how these vibrations interact with each other.
Instrumentation
Introduction
In this look at instrumentation for IR spectroscopy, we will be limiting our attention to
instrumentation concerned with spectroscopy in the middle region (4000 - 200cm-1). It is absorption
in this region which gives structural information about a compound.
Instrumental components
Sources
An inert solid is electrically heated to a temperature in the range 1500-2000 K. The heated material
will then emit infra red radiation.
The Nernst glower is a cylinder (1-2 mm diameter, approximately 20 mm long) of rare earth oxides.
Platinum wires are sealed to the ends, and a current passed through the cylinder. The Nernst glower
can reach temperatures of 2200 K.
The Globar source is a silicon carbide rod (5mm diameter, 50mm long) which is electrically heated
to about 1500 K. Water cooling of the electrical contacts is needed to prevent arcing. The spectral
output is comparable with the Nernst glower, execept at short wavelengths (less than 5 m) where
it's output becomes larger.
The incandescent wire source is a tightly wound coil of nichrome wire, electrically heated to 1100
K. It produces a lower intensity of radiation than the Nernst or Globar sources, but has a longer
working life.
Detectors
There are three catagories of detector;
" Thermal
" Pyroelectric
" Photoconducting
Thermocouples consist of a pair of junctions of different metals; for example, two pieces of bismuth
fused to either end of a piece of antimony. The potential difference (voltage) between the junctions
changes according to the difference in temperature between the junctions
Pyroelectric detectors are made from a single crystalline wafer of a pyroelectric material, such as
triglycerine sulphate. The properties of a pyroelectric material are such that when an electric field is
applied across it, electric polarisation occurs (this happens in any dielectric material). In a
pyroelectric material, when the field is removed, the polarisation persists. The degree of polarisation
is temperature dependant. So, by sandwiching the pyroelectric material between two electrodes, a
temperature dependant capacitor is made. The heating effect of incident IR radiation causes a
change in the capacitance of the material. Pyroelectric detectors have a fast response time. They are
used in most Fourier transform IR instruments.
Photoelectric detectors such as the mercury cadmium telluride detector comprise a film of
semiconducting material deposited on a glass surface, sealed in an evacuated envelope. Absorption
of IR promotes nonconducting valence electrons to a higher, conducting, state. The electrical
resistance of the semiconductor decreases. These detectors have better response characteristics than
pyroelectric detectors and are used in FT-IR instruments - particularly in GC - FT-IR.
Types of instrument
Dispersive infra red spectophotometers
These are often double-beam recording instruments, employing diffraction gratings for dispersion
of radiation.
Radiation from the source is flicked between the reference and sample paths. Often, an optical null
system is used. This is when the detector only responds if the intensity of the two beams is unequal.
If the intensities are unequal, a light attenuator restores equality by moving in or out of the reference
beam. The recording pen is attached to this attenuator.
Fourier-transform spectrometers
Any waveform can be shown in one of two ways; either in frequency domain or time domain.
Dispersive IR instruments operate in the frequency domain. There are, however, advantages to be
gained from measurement in the time domain followed by computer transformation into the
frequency domain.
If we wished to record a trace in the time domain, it could be possible to do so by allowing radiation
to fall on a detector and recording its response over time. In practice, no detector can respond
quickly enough (the radiation has a frequency greater than 1014 Hz). This problem can be solved by
using interference to modulate the i.r. signal at a detectable frequency. The Michelson
interferometer is used to produce a new signal of a much lower frequency which contains the same
information as the original IR signal. The output from the interferometer is an interferogram.
The Michelson interferometer
Radiation leaves the source and is split. Half is reflected to a stationary mirror and then back to the
splitter. This radiation has travelled a fixed distance. The other half of the radiation from the source
passes through the splitter and is reflected back by a movable mirror. Therefore, the path length of
this beam is variable. The two reflected beams recombine at the splitter, and they interfere (e.g. for
any one wavelength, interference will be constructive if the difference in path lengths is an exact
multiple of the wavelength. If the difference in path lengths is half the wavelength then destructive
interference will result). If the movable mirror moves away from the beam splitter at a constant
speed, radiation reaching the detector goes through a steady sequence of maxima and minima as the
interference alternates between constructive and destructive phases.
If monochromatic IR radiation of frequency, f ( ir ) enters the interferometer, then the output
frequency, fm can be found by;
where v is the speed of mirror travel in mm/s
Because all wavelengths emitted by the source are present, the interferogram is extremely
complicated.
The moving mirror must travel smoothly; a frictionless bearing is used with electromagnetic drive.
The position of the mirror is measured by a laser shining on a corner of the mirror. A simple sine
wave interference paatern is produced. Each peak indicates mirror travel of one half the wavelength
of the laser. The accuracy of this measurement system means that the IR frequency scale is accurate
and precise.
In the FT-IR instrument, the sample is placed between the output of the interferometer and the
detector. The sample absorbs radiation of particular wavelengths. Therefore, the interferogram
contains the spectrum of the source minus the spectrum of the sample. An interferogram of a
reference (sample cell and solvent) is needed to obtain the spectrum of the sample.
After an interferogram has been collected, a computer performs a Fast Fourier Transform, which
results in a frequency domain trace (i.e intensity vs. wavenumber) that we all know and love.
The detector used in an FT-IR instrument must respond quickly because intensity changes are rapid
(the moving mirror moves quickly). Pyroelectric detectors or liquid nitrogen cooled photon
detectors must be used. Thermal detectors are too slow.
To acheive a good signal to noise ratio, many interferograms are obtained and then averaged. This
can be done in less time than it would take a dipersive instrument to record one scan.
Advantages of Fourier transform IR over dispersive IR;
" Improved frequency resolution
" Improved frequency reproducibility (older dispersive instruments must be recalibrated for
each session of use)
" Higher energy throughput
" Faster operation
" Computer based (allowing storage of spectra and facilities for processing spectra)
" Easily adapted for remote use (such as diverting the beam to pass through an external cell
and detector, as in GC - FT-IR)
Review your learning
You should be aware of the various sources and detectors used in IR spectroscopy. You should also
have an understanding of how a Fourier transform instrument operates (including the
interferometer), and the advantages that this kind of instrument gives over the dispersive type.